Biology Microscope Quiz

Histology Practical - Microscope 2024

• Histology is the microscopic study of normal cells, tissues, and organs. Examination involves sectioning, staining, and viewing under a microscope.

Introduction

• Microscopes magnify small objects, allowing observation by the naked eye.
• Most microscopes use powerful lenses to enlarge specimens over 100 times their actual size.
• Microscopes are expensive, so proper handling is crucial.

Types of Microscopes

• Light (optical) microscopes use light to pass through a sample and create an image.
• Electron microscopes use beams of accelerated electrons for higher resolution viewing, revealing structures smaller than those visible with light microscopes. They have two types : transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

Parts of a Light Microscope

• Body tube: Includes the ocular (eyepiece) and revolving nosepiece with objective lenses.
• Arm: Used for carrying the microscope. It supports the rest of the microscope and has controls for coarse and fine focus adjustment.
• Stage: Supports the microscope slide holding the specimen; often has clips to hold the slide in place.
• Light source: Provides the light for viewing the specimen. Light should be mild to moderate.
• Condenser: Focuses and controls the light intensity directed onto the specimen.

Microscopy Diagram Labeling

• Key components of the light microscope, like stage clips, revolving nosepiece, ocular lens, arm, light source, body tube, base, and objective lenses are labeled on a diagram.

Electron Microscope

• Uses a beam of accelerated electrons as illumination.
• The short wavelength of electrons produces a higher resolving power than light microscopes.
• Enables examination of the structure of smaller objects.

Comparison Between Light & Electron Microscopes

• Light Microscope (LM): Uses visible light (often electric or transmitted daylight); non-vacuumed tube; view using glass lenses; magnification up to 1000 times & resolution around 0.2 µm; observations can be made by human eyes.
• Electron Microscope (EM): Uses invisible electron beam; vacuumed tube; magnification up to 100,000 times or more; resolution is around 0.003 micrometers (nm); view using electromagnetic coils; observations view by Fluorescent screen.

Tissue Preparation for Light Microscopy

• Several methods prepare specimens for microscopic examination:
  • Filming: Tissue fluids (e.g., blood films).
  • Teasing/Squeezing: Tissue structures such as muscle and nerves.
  • Stretching: Elastic tissue (e.g., fascia, membranes).
  • Sectioning/Cutting: The most common method, involves thin sections using a microtome (instrument for cutting slices). Often employing paraffin techniques.

Preparation of Paraffin Sections

• Obtaining the tissue: Must be fresh, with minimal thickness (less than 60 cm³) for good fixation.
• Fixation: Preserves the tissue in a life-like state to prevent postmortem changes (e.g., decomposition).
• Functions of Fixation:
  • Keeps proteins stable.
  • Prevents postmortem changes.
  • Reduces shrinkage and distortion.
  • Improves stainability by altering refractive indices.
• Fixatives: Common fixatives include 10% formalin or formal saline, Bouin's solution, osmic acid, glutaraldehyde, and combinations of alcohols.
• Wash excess fixative: Removing excess fixative reagent by running water or alcohol solutions.
• Dehydration: Gradually removing water from tissue using increasing concentrations of ethyl alcohol (e.g., 70%, 90%, 95%, 100%). This prevents shrinkage.
• Time needed for dehydration: Based on tissue thickness (<60 cm³), it takes 2-3 hours, and changes are done hourly, while absolute alcohol requires 1-1 hour each. The concentration of dehydrating agent should be around 10 times that of the tissue
• Dehydration effects: Over-dehydration leads to tissue hardening and a cloudy appearance. Incomplete dehydration results in tissue containing water and cracking.
• Clearing: Replacing alcohol with a solvent (e.g., xylol, methylbenzoate) that is miscible with paraffin. This makes the tissue ready for paraffin infiltration.
• Impregnation: Immerse the cleared tissue in melted paraffin to infiltrate and support the tissue. The process typically uses different paraffin baths and takes 1-2 hours per stage.
• Embedding: Embedding the tissue in paraffin to create a solid block for sectioning. Utilizing a paper or special metal molds.
• Frozen section: A method for preparing fresh, unfixed tissue for sections, often used in histochemical methods.

Precautions During Paraffin Section Preparation

• Handle specimens gently.
• Use an appropriate preparation schedule.
• Use high-quality paraffin wax.
• Avoid cross-contamination between tissue sections.

Staining

• Stain: A soluble coloring agent penetrating and retaining inside the tissue.
• Functions of stains:
  • Provides clearer observation and analysis of cells.
  • Identifies cell abnormalities.
  • Changes cell and tissue color significantly.
• Types of stains:
  • General stains: (e.g., hematoxylin and eosin, H&E).
  • Special stains: Reveal specific tissue components (e.g., Sudan black stains lipids).
  • Histochemical and cytochemical stains: Identify specific chemical components (e.g., PAS reaction for carbohydrates).

Steps of Staining Paraffin Sections using H&E Stain

• Deparaffinization (xylol): Removing paraffin.
• Hydration (alcohol): Returning to aqueous solutions.
• Staining (hematoxylin): Staining nuclei.
• Washing (running water): Removing excess stain.
• Counterstaining (eosin): Staining cytoplasm.
• Mounting (with a coverslip and mounting medium): Protecting the slide for examination.

Affinity to General Stain

• Basophilic: Acidic cell components (e.g., nucleus) have an affinity towards basic stains.
• Acidophilic: Basic cell components (e.g., cytoplasm) have an affinity towards acidic stains.
• Neutralophilic: Some components have affinity towards both acidic and basic stains.

Staining Methods & Alcohol Concentration

• Various alcohol concentrations are used during tissue preparation to prevent shrinkage and maintain tissue integrity during staining.

Steps of preparing paraffin sections

• A diagram summarizing the steps (fixation, dehydration, clearing, infiltration, embedding) in preparing a paraffin section.

Ground Preparation

• Technique for preparing thin sections of bone or tissue involving precise sawing, embedding in resin, grinding, and polishing.